YAP1 regulates chondrogenic differentiation of ATDC5 promoted by temporary TNF-α stimulation through AMPK signaling pathway

Molecular and Cellular Biochemistry - Local injection of tumor necrosis factor-alpha (TNF-α) at bone fracture sites during the early stage of the inflammatory response is reported to improve...     

Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G₂/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G₂/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.     

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.     

Effect of 5′-methylthioadenosine on induction of murine erythroleukemia cell differentiation

The polyamines putrescine, spermidine and spermine have been implicated in the regulation of proliferation and differentiation. The present study has monitored the effects of 5′-methylthioadenosine, the metabolic product of spermidine and spermine synthesis, on the appearance of a differentiated murine erythroleukemia cell phenotype. The results demonstrate that increasing concentrations of 5′-methylthioadenosine (1 × 10^?6 to 5 × 10^?4M) progressively inhibit murine erythroleukemia cell heme synthesis and hemoglobin production. The results also demonstrate that this inhibition of differentiation is not related to depletion of intracellular spermidine or cytostasis. Since 5′-methylthioadenosine is also a known inhibitor of DNA methylation, this naturally occurring nucleoside may be an intermediate involved in both murine erythroleukemia cell proliferation and differentiation.     

Significant increase in Young׳s modulus of ATDC5 cells during chondrogenic differentiation induced by PAMPS/PDMAAm double-network gel: Comparison with induction by insulin

A double-network (DN) gel, which was composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly(N,N′-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated the biomechanical and biological responses of chondrogenic progenitor ATDC5 cells cultured on the DN gel. ATDC5 cells were cultured on a polystyrene surface without insulin (Culture 1) and with insulin (Culture 2), and on the DN gel without insulin (Culture 3). The cultured cells were evaluated using micropipette aspiration for cell Young?s modulus and qPCR for gene expression of chondrogenic and actin organization markers on days 3, 7 and 14. On day 3, the cells in Culture 3 formed nodules, in which the cells exhibited an actin cortical layer inside them, and gene expression of type-II collagen, aggrecan, and SOX9 was significantly higher in Culture 3 than Cultures 1 and 2 (p<0.05). Young?s modulus in Culture 3 was significantly higher than that in Culture 1 throughout the testing period (p<0.05) and that in Culture 2 on day 14 (p<0.01). There was continuous expression of actin organization markers in Culture 3. This study highlights that the cells on the DN gel increased the modulus and mRNA expression of chondrogenic markers at an earlier time point with a greater magnitude compared to those on the polystyrene surface with insulin. This study also demonstrates a possible strong interrelation among alteration of cell mechanical properties, changes in actin organization and the induction of chondrogenic differentiation.     

MicroRNA-344 inhibits 3T3-L1 cell differentiation via targeting GSK3β of Wnt/β-catenin signaling pathway

Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of complex events in which microRNAs might play an essential role. In this study, we found that the overexpression of microRNA-344 (miR-344) inhibits 3T3-L1 cell differentiation and decreases triglyceride accumulation after MDI stimulation. We demonstrated that miR-344 directly targets the 3′ UTR of GSK3β (Glycogen synthase kinase 3 beta). Knockdown of GSK3β with siRNA results in inhibiting 3T3-L1 differentiation, while its overexpression restores the effect of miR-344. In addition, miR-344 elevates the level of active β-catenin, which is the downstream effector of GSK3β in the Wnt/β-catenin signaling pathway. These data indicate that miR-344 inhibits adipocyte differentiation via targeting GSK3β and subsequently activating the Wnt/β-catenin signaling pathway.     

14-3-3ζ binds class III phosphatidylinositol-3-kinase and inhibits autophagy

Autophagy is an evolutionarily conserved pathway involved in a great variety of physiological roles such as starvation adaptation, elimination of microorganisms, and intracellular protein and organelle clearance. It seems clear that autophagy is strictly controlled because of the multiplicity of its functions and thus, autophagy components are finely regulated. Here, 14-3-3ζ protein has been shown as negative regulator of autophagy by association and inactivation of the vesicle nucleation component vacuolar protein sorting 34 (hVps34), the class III phosphatidylinositol-3- kinase (PI3KC3).     

Down-regulation of β3-integrin inhibits bone metastasis of small cell lung cancer

Bone is one of the most frequent targets of small cell lung cancer (SCLC) metastasis, but the molecular mechanism remains unclear. β3-integrin plays an important role in invasion of various kinds of tumors. Yet, its role in bone-metastasis of SCLC is still unknown. In this study, we first examined the expression of β3-integrin in SBC-5 and SBC-3 cells by real-time PCR, western blot and immunofluorescence. We found that, compared to none bone-metastatic SBC-3 cells, β3-integrin was highly expressed in SBC-5 cells, a specific bone-metastatic SCLC cells line characterized in our previous study. We next constructed β3-integrin siRNA and transfected SBC-5 cell line, and found that β3-integrin siRNA significantly down-regulated the β3-integrin mRNA level and protein expression in SBC-5 cell line. We further found that inhibition of β3-integrin significantly reduced tumor cell proliferation and induced apoptosis. In addition, the β3-integrin down-regulated cells presented significant decrease in cell adhesion, migration and invasion activity. Our results suggest the β3-integrin has an essential effect on tumor cell proliferation and progression, and may be a potential therapeutic target for the prevention of skeletal metastases of lung cancer.     

Myostatin inhibits brown adipocyte differentiation via regulation of Smad3-mediated β-catenin stabilization

Brown adipocytes play an important role in regulating energy balance, and there is a good correlation between obesity and the amount of brown adipose tissue. Although the molecular mechanism of white adipocyte differentiation has been well characterized, brown adipogenesis has not been studied extensively. Moreover, extracellular factors that regulate brown adipogenic differentiation are not fully understood. Here, we assessed the mechanism of the regulatory action of myostatin in brown adipogenic differentiation using primary brown preadipocytes. Our results clearly showed that differentiation of brown adipocytes was significantly inhibited by myostatin treatment. In addition, myostatin-induced suppression of brown adipogenesis was observed during the early phase of differentiation. Myostatin induced the phosphorylation of Smad3, which led to increased β-catenin stabilization. These effects were blocked by treatment with a Smad3 inhibitor. Expression of brown adipocyte-related genes, such as PPAR-γ, UCP-1, PGC-1α, and PRDM16, were dramatically down-regulated by treatment with myostatin, and further down-regulated by co-treatment with a β-catenin activator. Taken together, the present study demonstrated that myostatin is a potent negative regulator of brown adipogenic differentiation by modulation of Smad3-induced β-catenin stabilization. Our findings suggest that myostatin could be used as an extracellular factor in the control of brown adipocyte differentiation.     

Protein kinase C-θ inhibits inducible regulatory T cell differentiation via an AKT-Foxo1/3a-dependent pathway

Protein kinase C (PKC)-θ has been shown to be a critical TCR signaling molecule that promotes the activation and differentiation of naive T cells into inflammatory effector T cells. In this study, we demonstrate that PKC-θ-mediated signals inhibit inducible regulatory T cell (iTreg) differentiation via an AKT-Foxo1/3A pathway. TGF-β-induced iTreg differentiation was enhanced in PKC-θ(-/-) T cells or wild-type cells treated with a specific PKC-θ inhibitor, but was inhibited by the PKC-θ activator PMA, or by CD28 crosslinking, which enhances PKC-θ activation. PKC-θ(-/-) T cells had reduced activity of the AKT kinase, and the expression of a constitutively active form of AKT in PKC-θ(-/-) T cells restored the ability to inhibit iTreg differentiation. Furthermore, knockdown or overexpression of the AKT downstream targets Foxo1 and Foxo3a was found to inhibit or promote iTreg differentiation in PKC-θ(-/-) T cells accordingly, indicating that the AKT-Foxo1/3A pathway is responsible for the inhibition of iTreg differentiation of iTregs downstream of PKC-θ. We conclude that PKC-θ is able to control T cell-mediated immune responses by shifting the balance between the differentiation of effector T cells and inhibitory Tregs.